Te inverted microscope

Cells (4 × 10⁴, 50% confluency) were starved for 1 h in a reduced serum medium, plated onto a fibronectin-coated (10 μg/ml) 96-well plate, fixed in 4% paraformaldehyde, and stained with crystal violet. Cell adhesion was quantified by absorbance at 570 nm. For analysis of cell spreading, cells seeded onto an Ibidi 8-well μ-Slide were imaged with a Nikon TE inverted microscope after 24 h, and the cell surface was quantified by ImageJ. Tumor cell invasion across Matrigel-coated 8-μm PET Transwell chambers (Corning) was quantified in the presence of an NIH3T3-conditioned medium as a chemoattractant (26 ). The 3D organotypic tumor spheroids embedded in a collagen matrix were quantified for maximum invasion distance and invasion area (38 (link)).

Cell migration was assessed by wound-healing scratch assay as previously described59 . After isolation, cardiac fibroblasts (∼3 × 10⁵) were plated in 12-well tissue culture plates. Twenty-four hours after plating, scratches were made using 100 μl pipette tips and the wells were washed twice with PBS. Then the cells were stimulated with conditioned media from NRVMs stimulated with aldosterone (1 μM) for 24 h. Following 3 and 12 h of stimulation, the cells were fixed in 3.7% paraformaldehyde and stained with 0.1% crystal violet staining solution. Photographs were taken on Nikon TE inverted microscope connected to a Nikon camera. Quantification of cell migration was performed by measuring the distance between 10 random points within the wound edge. Gap distance of the wound was measured using ImageJ software, and the data were normalized to the average of the wound of control cells fixed at the time of scratches.